R e a l-T im e a na lysis o f R na i Du p l e x e s

Sean M. McCarthy and Martin Gilar Waters Corporation, Milford, MA, U.S.A.

INT RODUCT ION

RNA interference (RNAi) is a rapidly emerging strategy for temporar-

ily silencing genes and preventing protein translation. RNAi is a

double-stranded non-coding RNA molecule designed to bind to a

specific mRNA target, and via a cascade of biochemical reactions

interfere with the protein production. This method of gene silencing is

currently being utilized in a variety of animal studies and is receiving

increased of attention as a potential therapeutic strategy for humans.

A major challenge for developing human therapeutics preventing

protein translation remains the assurance of RNAi purity. The

presence of certain related impurities may lead to the possibility of

unwanted, and perhaps detrimental, non-targeted gene silencing.

Major sources of impurities in RNAi duplexes are often the result

of degradation, intra-molecular hybridization mismatches, or

more commonly incomplete syntheses of the complementary

single-stranded RNA complementary strands. The presence of

non-hybridized single stranded RNAi is also undesirable and often

associated with a decrease in therapeutic potency.

In this application note, we describe a rapid and highly sensitive

method for the routine analysis of RNAi duplexes preventing

protein translation using the Waters® ACQUITY UPLC® System with

Oligonucleotide Separation Technology (OST) Column chemistry.

MET HODS

LC conditions LC system: Waters ACQUITY UPLC® System

Column: ACQUITY UPLC OST C18 2.1 x 50 mm, 1.7 µm

Column temp.: 20 °C

Flow rate: 0.2 mL/min

Mobile phase A: 0.1 M TEAA, pH 7.5

Mobile phase B: 20% Acetonitrile in A

Gradient: 35 to 85% B in 10.0 min (1% ACN/min)

Detection: ACQUITY UPLC PDA, 260 nm

Sample

RNAi complementary strands 5’ - UCG UCA AGC GAU UAC AAG

GTT - 3’ (upper) and 5’ - CCU UGU AAU CGC UUG ACG ATT - 3’

(lower) were purchased from Integrated DNA Technologies and

reconstituted in 110 µL of 0.1 M triethylammonium acetate (TEAA)

to yield concentrations of approximately 2.5 nmol/µL. The samples

were purified prior to use1 and purity was verified prior to duplex

formation experiments (Figure 1).

Figure 1. Determination of single-stranded RNA oligonucleotide purity. (* = non-oligo impurity.)

RESULTS AND DISCUSSION

RNAi duplex formation

RNAi duplexes were prepared by combining appropriate molar ratios

of upper and lower (2:1 and 1:2) complementary strands in 0.1 M

TEAA. Mixtures were heated at 90 °C for 5 minutes and gradually

cooled to 20 °C. Samples were prepared immediately prior to use to

minimize sample degradation.

0 8Minutes

Singe Stranded Lower

Single Stranded Upper

*

UPLC conditions

RNAi duplex samples were separated on a Waters ACQUITY UPLC

System using an ACQUITY UPLC OST C18 2.1 x 50 mm, 1.7 µm

column using ion-pairing reversed phase chromatography.2

Separated RNA species were detected with an ACQUITY UPLC PDA

detector scanning from 19 to 350 nm.

As shown in Figure 2, this system offered exemplary component

resolution with no evidence of on-column duplex degradation or

melting. Additionally, this method offers impressive separation of

the desired duplex from impurities present in the sample, primarily

mismatched sequences.

In the presence of excess single-stranded RNA, retention times of

both the excess single-stranded RNA and duplex remain constant,

highlighting the utility of this method for purification of RNAi. The

method also allows for the separation of failure sequence and other

mismatch duplexes from the desired duplex product.

Figure 2. Separation of RNAi duplex from excess single-stranded upper (panel A) and lower (panel B) RNA. (* = non-oligo impurity.)

To fully investigate the utility of the chromatographic system and

to confirm that there was no on-column melting of RNA duplexes

with this method, we investigated whether our separation conditions

would allow for the formation of RNA duplexes on an ACQUITY

UPLC OST Column. To accomplish this, we separately injected each

of the complementary RNAi strands under 100% binding conditions

followed by gradient elution of the bound samples.

We found quantitative formation of an RNAi duplex following injec-

tion of upper and lower RNAi strands, with the resulting duplex peak

exhibiting the identical retention time of authentic RNAi prepared

via a separate annealing step (Figure 3). This data strongly indicates

that under our UPLC separation conditions RNAi duplex does not melt

during LC separation. In fact, it appears that spontaneous on-column

annealing is favored as is indicated by the appearance of only one

single-stranded peak present in a duplex/single-strand mixture.

Once quantitative duplex formation is accomplished, the duplex

peak is predominant and well-separated from other impurities.

Figure 3. RNAi duplex formation on an ACQUITY UPLC OST Column. Single-stranded upper loaded on column at 35% B and single-stranded lower loaded immediately after. (* = non-oligo impurity.)

0 8Minutes

Single StrandedUpper

Duplex

MismatchDuplexes

Single StrandedLower

Duplex

*

MismatchDuplexes

A

B

0 8Minutes

Duplex

Single StrandedUpper

*

MismatchDuplexes

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

CONCLUSIONS

The data presented highlights the superior chromatographic resolu-

tion possible using the ACQUITY UPLC System and OST Column

chemistry. This UPLC system solution offers superior performance

for the efficient detection, quantification, and chromatographic

resolution of RNAi duplexes from their single-stranded and

mismatched counterparts, and also shows considerable utility

in monitoring their formation.

The described UPLC method enables rapid and real-time quality con-

trol analysis of the reaction progress, eliminating the excessive RNAi

characterization times associated with other analytical methods.

This method does not effect additional degradation of the duplex,

enabling accurate, consistent, and reproducible analysis of RNAi

duplexes. Finally, the UPLC method offers superior resolution, allow-

ing for the detection and quantitation of excess single-stranded

RNA, mismatched duplexes, and synthetic impurities.

Overall, this enables the direct analysis of reaction products and

allows for purity determination in a high throughput manner.

References

1. Semi-Preparative Scale Single Stranded RNA Purification. Waters Application Note. 2008: 720002602en.

2. UPLC/MS Analysis of Interfering RNA Oligonucleotides. Waters Application Note. 2008: 720002412en.

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